Method and kit for monitoring mammalian reproductive cycles

ABSTRACT

A kit for monitoring mammalian reproductive cycles by monitoring variations in the quantity of one or more low molecular weight volatile compound having a molecular weight of less than 50 grams per mole present in a body constituent sample is disclosed. Samples of a body constituent selected from the group consisting essentially of humoral fluid, breath and body cavity air are collected from a female mammal a multiple number of times during the reproductive cycle. The quantity of a low molecular weight volatile compound in each sample is measured. In the preferred embodiment, the low molecular weight volatile compound, acetaldehyde, will be measured and monitored. Variations in the quantity of the low molecular weight volatile compound appearing in each sample is monitored to determine the phase of the mammal&#39;s reproductive cycle and to predict the occurrence of ovulation.

ORIGIN OF THE INVENTION

The invention described herein was made using federal funds and theUnited States Department of Agriculture shall have the non-exclusiveright to practice the invention for government purposes on behalf of theUnited States throughout the world.

This application is a divisional application of U.S. Ser. No.08/293,666, "Method and Kit for Monitoring Mammalian ReproductiveCycles," filed Aug. 22, 1994 now U.S. Pat. No. 5,721,142.

FIELD OF THE INVENTION

The invention relates to monitoring variations in the quantity of lowmolecular weight volatile compounds present in body constituents ofmammals to determine phases of the reproductive cycle and to predict theoccurrence of ovulation. The invention also relates to monitoringvariations in the quantity of low molecular weight volatile compoundspresent in body constituents of animals to detect the onset of estrusand predict the occurrence of ovulation.

BACKGROUND OF THE INVENTION

Monitoring reproductive cycles and predicting the time of ovulation inmammals is of great importance to human reproduction and the productionof livestock and other animals. Means currently available for detectingovulation, however, have considerable limitations. For example, surgicaltechniques for detecting ovulation require that incisions be made so thecorpus luteum of the ovary can be observed for physical signs ofovulation. Such a procedure is undesirable and has not gained widespreadacceptance. Moreover, clinical evaluations , such as monitoring pelvicdiscomfort or monitoring basal body temperature are not widely acceptedbecause of the imprecision of the methods and their unreliability forpredicting ovulation.

Various biochemical and histological methods for detecting ovulation arealso available. Cyclic variations in the concentrations of certainhormones appearing in the blood, such as rises in serum estrogen with arise in luteinizing hormone, are known indicators of impending ovulationin humans. Measuring the glucose concentration in cervical mucosa andmeasuring salivary alkaline phosphatase levels have also been exploredas methods for detecting ovulation. Because of the risk of samplecontamination and the amount of technical expertise required toaccurately perform necessary collections and analyses, histological andbiochemical tests for predicting the occurrence of ovulation oftenrequire trained personnel to perform the procedures. Many of thesemethods, however, remain unreliable in predicting the onset of thefertile period or the occurrence of ovulation.

Vaginal secretions have been monitored for the concentration of volatileorganic compounds having a molecular weight between 50 and 350 grams permole for use as predictors of the fertile period and ovulation. U.S.Pat. No. 3,986,494, "Method of Predicting and Detecting Ovulation",Preti et al., Oct. 19, 1976. The concentration of a particular volatileorganic compound, such as acetic or lactic acid, is used to diagnose theoccurrence of ovulation in the menstrual cycle. The compounds monitoredhave a first increase in concentration just prior to the rise in serumestrogens, thereby indicating the onset of the fertile period. At leastfour days after the first increase, a second increase in the volatileorganic compound indicates the time of ovulation. This method isestimated to be useful in accurately predicting the fertile period andovulation in approximately only 80% of the female human population.

U.S. Pat. No. 4,010,738, "Method of Predicting and Detecting Ovulation",Preti et al., Mar. 8, 1977, discloses monitoring urea concentrations invaginal secretions of mammals as a method of diagnosing the onset of thefertile period or ovulation. As with other methods known for monitoringvarious compounds in vaginal secretions, the likelihood of contaminationof the secretion with other body secretions or feces is great. Moreover,urea concentration is influenced by nutrition and digestion, and is nota reliable indicator of reproductive cycle events.

Other methods for detecting the onset of the fertile period andovulation include monitoring the volatile sulfur content of mouth air.U.S. Pat. No. 4,119,089, "Method of Predicting and Determining Ovulationby Monitoring the Concentration of Volatile Sulfur-Containing CompoundsPresent in Mouth Air", Preti et al., Oct. 10, 1978. The volatile sulfurcontent of mouth air is believed to be a secondary characteristic whichis responsive to elevated levels of female sex hormones. A first markedincrease in the concentration of volatile sulfur compounds after mensesis reported as being predictive of ovulation. A second marked increasein sulfur concentration is reported to be diagnostic of ovulation.Although detection of volatile sulfur content of mouth air may in someway be correlated to, or at least occurring at similar times withparticular periodontal conditions occurring at ovulation, the volatilesulfur content of mouth air may also be influenced by other systemicconditions. Thus, it may not be a reliable predictor of ovulation.

Methods for determining the occurrence of estrus in cattle have alsobeen disclosed. Direct rectal palpation or ultrasonography of theovaries can be performed, however, it is not a viable choice for use inthe field by farmers and dairymen. Similarly, measuring the pulsatilerelease of luteinizing hormone (LH) in serum is not a practical meansfor monitoring estrus by livestock producers. Other means for detectingestrus, such as serum or milk progesterone level measurement andelectronic conductivity tests of cervicovaginal mucus are not accurateand give only retrospective evaluation of the reproductive cycle.

Cow vaginal secretions may be collected over time to determine asignificant increase in the amounts of an indicator compound in thesecretions. U.S. Pat. No. 4,467,814, "Method for Detecting Bovine Estrusby Determining Methyl Heptanol Concentrations in Vaginal Secretions",Preti et al., Aug. 28, 1984. The indicator compounds are eight-carbonalcohols such as methyl-1-heptanols, particularly 6-methyl-1-heptanol.Specific quantities of the indicator compounds are reported asindicative of estrus. The high risk of contamination and the requirementthat specific quantities of compound be identified, as opposed tomonitoring variations in quantities, in order to predict estrus makesuch a method undesirable for monitoring estrus cycles.

Volatile compounds present in blood have been investigated for use asindicators of estrus. Klemm et al., Blood acetaldehyde fluctuatesmarkedly during bovine estrous cycle, In press, Anim. Reprod. Sci.;Klemm, W. R., Acetaldehyde As a Possible Marker and Predictor of BovineEstrus, In press, Beef Cattle Research in Texas. The low molecularweight compound acetaldehyde was found to increase a few days beforebehavioral signs of estrus and decrease markedly on the day of estrus orshortly thereafter. Methods for measuring and monitoring acetaldehydelevels in blood or other humoral fluids would allow estrus and/orovulation in mammals to be predicted.

There remains a great need for a simple, universally acceptable methodfor detecting and diagnosing mammalian reproductive cycle phases,particularly the occurrence of ovulation. While the shortcomings of themethods discussed apply for mammalian species; predicting ovulation inhuman females is even more difficult because there are not clearbehavioral signs that ovulation is about to occur.

Accurately identifying the time of ovulation in mammals willdramatically increase the likelihood that fertilization occurs andoffspring is produced. In cases of particular human medical concerns,such as infertility, diagnosing the time of ovulation is critical toconception. Accurately predicting ovulation will also enable developingreliable rhythm-type birth control methods for humans.

Predicting the occurrence of estrus and ovulation is economicallyimportant to livestock breeders, particularly cattle breeders. In orderto increase milk production in dairy cattle, and maximize offspring inboth dairy and beef cattle, detection of estrus is required. Detectingand predicting estrus and ovulation is particularly important in dairyherds, where artificial insemination is nearly exclusively used toproduce fertilization. Larger dairy herd sizes and rising labor costsfurther increase the need for a method for easily and accuratelydetecting estrus. Because bovine estrus (lordosis or standing matingbehavior) is short (1-18 hours, mean 4.4 hours), with ovulationoccurring at about 12 hours after the onset of estrus, there is a herdmanagement need to develop simple chemical tests for compounds thatcould serve as biochemical markers and predictors of estrus andovulation. Identification of one or more compounds in a readilyaccessible body constituent would be an important step in detectingbovine estrus. The common practice of visual monitoring and measuringblood progesterone as indexes of stage of estrus could then be replacedby a more accurate method for detecting estrus and ovulation.

SUMMARY OF THE INVENTION

The present invention is a method for monitoring mammalian reproductivecycles by monitoring variations in the quantity of one or more lowmolecular weight volatile compound subject to variation during thereproductive cycle present in a body constituent sample. Samples of abody constituent selected from the group consisting essentially ofhumoral fluid, breath and body cavity air, are collected from a femalemammal a multiple number of times during the reproductive cycle. Humoralfluid samples can be selected from the group consisting essentially ofblood, vaginal secretions, saliva, urine, milk, sweat, skin glandsecretions, follicular fluid, and the air above a humoral fluid. Thequantity of a low molecular weight volatile compound in the sample ismeasured by using head-space gas chromatography, a chemical reagenttest, electrochemical detector, or other technique known for measuringthe quantity of the low molecular weight volatile compound present. Thelow molecular weight volatile compound will have a molecular weight ofless than 50 grams per mole. In the preferred embodiment, the lowmolecular weight volatile compound, acetaldehyde, will be measured andmonitored. Variations in the quantity of the low molecular weightvolatile compound appearing in each sample is monitored to determine thephase of the mammal's reproductive cycle and to predict the occurrenceof ovulation.

The body constituent sample may be separated into a nonvolatilecompounds fraction and a volatile compounds fraction whereby thevolatile compound's fraction of the sample collected is analyzed. Breathand body cavity air samples may be collected from within the mouth orbody cavity, respectively, or from the outside of the mammal's body.

The body constituent samples may be taken from primates or non-primates.Variations in the quantity of low molecular weight volatile compound aremonitored in primates to predict the occurrence of ovulation, whereinthe low molecular weight volatile compound in the body constituentsharply increases over baseline levels prior to ovulation and thendecreases to approximately baseline levels near or at the time ofovulation. The quantity of low molecular weight volatile compound in thesample of the body constituent of non-primates sharply increases overbaseline levels before estrus and decreases at approximately estrus.Monitoring the variations in the quantity of low molecular weightvolatile compounds present in body constituent samples in nonprimateswill enable predicting the occurrence of ovulation and/or estrus.

Predicting estrus and ovulation in animals can be accomplished bymonitoring variations in the quantity of one or more low molecularweight volatile compound subject to variation during the reproductivecycle in a body constituent selected from the group consistingessentially of humoral fluid, breath and body cavity air. Humoral fluidcan be selected from the group consisting of blood, vaginal secretions,saliva, urine, milk, sweat, vulval, skin gland secretions, follicularfluid and the air above a humoral fluid. The low molecular weightcompound measured in the body constituent samples will have a molecularweight of less than 50 grams per mole. In the preferred embodiment, thelow molecular weight volatile compound, acetaldehyde, is measured andvariations monitored. The samples are collected a preselected number oftimes during proestrus and the quantity of low molecular weight volatilecompound in each sample measured. Monitoring variations in the quantityof the low molecular weight volatile compound can be used to predict theonset of estrus and ovulation. The quantity of low molecular weightvolatile compound appearing in the body constituent sample will sharplyincrease over baseline levels shortly before estrus and decrease atapproximately the onset of estrus whereby the occurrence of ovulationcan then be predicted.

Samples of breath or body cavity air may be taken from the mouth or thebody cavity, respectively, or from outside the animal's body. Thequantity of low molecular weight volatile compound in a body constituentsample may be measured using head-space gas chromatography, abiochemical reagent test or other technique known for measuring thequantity of the low molecular weight volatile compound present.

The occurrence of ovulation in animals in the field may be predicted bymonitoring variations in the quantity of the low molecular weightvolatile compound in a body constituent during the reproductive cycle.The quantity of low molecular weight compound in the body constituentmay be measured using a chemical reagent test. A kit is providedcomprising an air adsorption tube containing an adsorbent with which thelow molecular weight volatile compound reacts. A pump having a flowcontrol meter will draw a body constituent sample through the airadsorption tube at a calibrated rate so as to expose the air to theadsorbent, thereby trapping the low molecular weight volatile compound.In the preferred embodiment, 1-(hydroxymethyl) piperidine is theadsorbent. In an alternate embodiment, di-nitrophenyl hydrazine may beused as the adsorbent. In yet another embodiment, 1, 3-cyclohexanedionemay be used as the adsorbent. The quantity of the low molecular weightvolatile compound is measured using ultraviolet spectroscopy techniques.

The quantity of low molecular weight compound in the body constituentmay also be measured using a kit comprising an electrochemical detectorand means for signalling a change in the quantity of the low molecularweight volatile compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gas chromatogram of profiles of blood volatiles related toestrus (Day 0).

FIG. 2a is a gas chromatogram of the variations in quantity ofacetaldehyde in blood head space during four estrous cycles in the samecow.

FIG. 2b is a gas chromatogram of the variations in quantity ofacetaldehyde in blood head space during four estrous cycles in the samecow.

FIG. 2c is a gas chromatogram of the variations in quantity ofacetaldehyde in blood head space during four estrous cycles in the samecow.

FIG. 3a is a gas chromatogram of the variations in quantity ofacetaldehyde in blood head space during three estrous cycles in the samecow.

FIG. 3b is a gas chromatogram of the variations in quantity ofacetaldehyde in blood head space during three estrous cycles in the samecow.

FIG. 4a is a gas chromatogram of the effects of body constituent samplepreparation procedures.

FIG. 4b is a gas chromatogram of the effects of body constituent samplepreparation procedures.

FIG. 4c is a gas chromatogram of the effects of body constituent samplepreparation procedures.

FIG. 5a is a mass spectra of blood head space derivatized with DNPH.

FIG. 5b is a complete chromatographic profile appearing before estrus.

FIG. 6a is a gas chromatogram of the variations in quantity ofacetaldehyde in milk head space during an estrous cycle in a cow.

FIG. 6b is a gas chromatogram of the variations in quantity ofacetaldehyde in milk head space during an estrous cycle in a cow.

FIG. 6c is a gas chromatogram of the variations in quantity ofacetaldehyde in milk head space during an estrous cycle in a cow.

FIG. 6d is a gas chromatogram of the variations in quantity ofacetaldehyde in milk head space during an estrous cycle in a cow.

FIG. 7 is a graph of the quantity of a acetaldehyde present in bovinevaginal air of three cows at estrus.

FIG. 8 is a kit for collecting body cavity air samples.

FIG. 9 is a kit for collecting samples of air from above a humoralfluid.

DETAILED DESCRIPTION

Body constituents, such as humoral fluids, breath and body cavity air,may be collected multiple times over a predetermined period of time frommammals and analyzed to measure the quantity of a low molecular weightvolatile compound or compounds subject to variation during thereproductive cycle present in the body constituent. Variations in thequantity of low molecular weight volatile compound measured is used formonitoring the mammal's reproductive cycles and to predict ovulation.Humoral fluids include blood, vaginal secretions, saliva, urine, milk,sweat, skin gland secretions, follicular fluid and the air above thehumoral fluid. For example, the air above or adjacent to a milk sampleor milk in bulk, can be analyzed to measure the quantity of lowmolecular weight volatile compound present. Body cavity air may besampled from the lungs and reproductive tract, as well as other bodycavities.

The body constituents can be used as a source of one or more lowmolecular weight volatile compounds, which provides a chemical signalfor determining and predicting the occurrence of ovulation and otherreproductive cycle events in mammals. Although one or more bodysubstance may be sampled and analyzed for one or more low molecularweight compounds to monitor mammalian reproductive cycles, for purposesof this discussion both body constituent and low molecular weightcompound will be discussed in the singular form.

In the preferred embodiment of the present invention, body constituentsamples are collected from a selected mammal or group of mammalsmultiple times during a reproductive cycle. Samples of humoral fluids,breath and body cavity air may be taken directly, using samplingprotocols known to those skilled in the art. The samples are thenanalyzed to monitor variations in the quantity of low molecular weightcompound present. Variations in the quantity of low molecular weightcompound can be used to monitor and predict the occurrence of ovulationand/or estrus. In the alternative, the air emitting from the body cavityof a mammal, such as the air external to a mammal's vagina orreproductive cavity at the vulva, may be sampled. As anotheralternative, samples of the air above a humoral fluid can be collectedfor analysis.

The body constituent samples are analyzed to measure the quantity of thelow molecular weight volatile compound in the sample. Low molecularweight volatile compound is used herein to refer to small, volatileorganic compounds having a molecular weight of less than 50 grams permole. The quantity of a low molecular weight volatile compound may beexpressed as concentration, parts per million (ppm), or other standardquantitative expression, depending upon the body constituent sampled andthe analytical procedures used to measure the quantity of the compound.Monitoring the variations in the quantity of the low molecular weightcompound present in a sample can be used to determine and predict phasesof the reproductive cycle, such as the occurrence of ovulation. A sharpand sudden increase in the quantity of the low molecular weight volatilecompound followed by a sharp and sudden decrease in the quantity of thelow molecular weight volatile compound in the body constituent sampleindicates that the mammal is at or near ovulation and/or estrus. Bodyconstituents of mammals will contain a baseline (generally lower)quantity of low molecular weight compounds subject to variation duringthe reproductive cycle during the other phases of the cycle.

Body constituent samples may be analyzed using head-space gaschromatography, chemical reagent tests, or electrochemical detectors, aswell as other analytical tools such as biochemical, immunochemical andphotochemical methods for measuring the quantity of low molecular weightvolatile compound. In the preferred embodiment, body constituent samplesare analyzed using head-space gas chromatography following the protocoldescribed in Example I.

EXAMPLE I

Whole blood samples were collected during 18 estrous cycles from fivegroup-housed adult female cattle. The cattle were fed and watered adlibitum, and observed daily between 08:00 and 10:00 h for signs ofstanding heat behavior (mounting and standing for being mounted). Ifneeded, cows were also observed in late afternoons near the expectedtime of estrus. Some of the cycles were induced by hormone treatmentconsisting of two daily injections of follicle stimulating hormone (FSH)followed by an injection of prostaglandin F2 (PG) two days later. Atotal of 164 samples were analyzed according to the following head-spacegas chromatography protocol to measure the quantity of the low molecularweight volatile compound, acetaldehyde, having a molecular weight ofapproximately 44 grams per mole. The blood samples were collected inearly morning by venipuncture.

The samples were prepared by placing a 10 ml aliquot of blood with 10%sodium citrate in a 30 ml injection vial, which was sealed with ateflon-coated septum. The samples were rapidly frozen and stored at -80°C. until being prepared for analysis by gas chromatography. Foranalysis, the samples were thawed and warmed to 40° C., a temperaturesimilar to that of the body temperature of the animal. It is preferredthat the samples be handled at a temperature that does not exceed thebody temperature of the mammal.

Upon thawing of the samples, the air (vapor phase) above the whole bloodwill contain the volatile compounds. The vapor phase provides a naturalprocess for separating the volatile compounds fraction from thenonvolatile compounds fraction of the blood. Head-spacair-tight syringewith an air-tight syringe that has been heated to prevent carry-over ofvolatile compound which would otherwise condense or adsorb on theglassware. The injection of air must be small enough to achieve aninjection that is rapid enough to produce clearly resolved peaks. In thepreferred method, approximately 1 ml of air injected achieved a rapidinjection suitable for analyzing the volatile compounds.

The chromatographic temperature profile began at room temperature inorder to get retention and separation of the lower molecular weight,more volatile compounds. The blood head space was injected into thesplitless port of a Hewlett Packard HP-5890 gas chromatograph. A 10 mfused silica pre-column, in series with a 15 m HP-5 (Hewlett Packard)(0.53 mm I.D., film thickness 2.65 μm) and a 15 m BP-1 (SGE) (0.53 mmI.D., film thickness 5.0 μm) nonpolar columns were used. As analternative, a 30 m DB-1 nonpolar column (J & W Scientific, Folsom,Calif.) may be used.

Column temperature began at 30° C. and was held at that temperature for10 minutes, followed by an increase of 4° C. every minute until atemperature of 110° was reached. The injector temperature was 180° C.and flame ionization detector (FID) temperature was 200° C. The flowrate of the helium carrier gas was 4.1 ml min⁻¹. Peak areas werecalculated with the PC software "ChromPerfect" by Justice Innovations,Inc., Palo Alto, Calif.

Acetaldehyde was represented by peak 3, which can be seen as the thirdpeak in FIG. 1 at the arrows. Acetaldehyde was eluted in the first fewminutes at room temperature. The typical chromatographic profile, shownin FIG. 1, revealed an initial double peak followed by two clearlydistinguishable peaks. The peaks shown in FIG. 1 all eluted within 4minutes (when the column was still at room temperature). FIG. 1 showschromatograms of the same cow in various stages of the estrous cycle.Day 0 is the day when the cow stood for mounting, which is the typicalbehavioral sign of bovine estrus. Day 0 was confirmed by rectalpalpations of the ovaries. Acetaldehyde is shown at the arrows andincreases on days -3 (3 days before standing estrus) to -1 (one daybefore standing estrus). The plot is the FID response as a function oftime after injection of sample on the column.

In the first three cows studied in the spring and summer through fourcycles each, acetaldehyde increased several days before estrus and thendecreased as seen in FIGS. 2a, 2b, and 2c. FIGS. 2a, 2b and 2c show thechanges in acetaldehyde in blood head space (nmol⁻¹) during four estrouscycles in three cows: Cow Red, Cow Pink and Cow White, respectively. Inall cycles, the relative amounts of acetaldehyde increased and thensuddenly decreased at or near estrus. The size of the peak representingacetaldehyde varied widely during the cycle and among cows. However, allcows typically showed a similar qualitative pattern of a rise and thenfall in peak area at or near the time of estrus (the day when cows willstand still for mounting).

A "baseline" quantity of acetaldehyde appearing in samples was found,although the quantity varied from animal to animal. "N" signifies thatthe estrus was natural (not preceded by FSH and PG injections). Absolutevalues were calculated from aqueous solutions of authentic standards ofacetaldehyde. Acetaldehyde (peak 3) was also found to be correlated withthe sexual hormones estrogen and progesterone. It was discovered thatacetaldehyde increased the day or so before estrogen rise until the day(±1) of standing estrus, whereupon acetaldehyde decreased to low orundetectable levels.

Similarly, FIGS. 3a and 3b show the changes of acetaldehyde in bloodhead space during six estrous cycles in two other cows: Cow 89124 andCow 87086, respectively. As seen in FIGS. 2a, 2b and 2c, the relativeamounts of acetaldehyde again increased and then decreased at or nearestrus in all six cycles. The first estrus in both cows was triggered byinjection of FSH and PG. Similar results were observed for bothhormone-induced and natural ("N") estrus.

In all five cows the absolute amounts of acetaldehyde varied greatlyfrom cow to cow, but the qualitative patterns during estrous cycles weresimilar. Near the time of estrus, there was a sudden rise and then asharp fall of blood acetaldehyde just prior to standing estrus.

Acetaldehyde was confirmed as the low molecular weight volatile compoundby preliminary mass spectral analysis. The blood samples were spikedwith candidate compounds to determine which compound co-eluted with thepeak of interest. To collect sufficient quantity of the compound formass spectral identification, 10 ml aliquots of blood were subjected tothe following procedures were tested to determine whether the yield ofacetaldehyde in the head space could be increased:

1. Shaking the samples at 50° C. for 3 min with a Mistral Multi-mixer.

2. Heating samples to various temperatures, starting at 30° C. andincreasing by 10° C. up to 100° C.

3. Leaving samples in a 90° C. water bath for varying lengths of time,starting with 10 min and increasing by 10 min up to 60 min.

4. Samples with varied sample phase fraction (SPF), which refers to theratio of liquid to head-space volume in the sealed injection vial, wasvaried from 33% to 40%, and from there, in 10% increments to 90% SPF.

5. Samples had nanopure water added to their sealed vials, starting with2.5 ml and increasing by 2.5 ml up to 10 ml. The extra pressureaccumulated in the vials was not vented. The effect of reducing watercontent of the sample by adding a molecular sieve compound (60/80 mesh,Supelco, Bellefonte, Pa.) was also tested. After sealing a vialcontaining the 0-4 g of sieve, 10 ml of air was withdrawn nd the 10 mlof blood injected. Samples were frozen and later tested under thestandard conditions of standing for 30 minutes at 90° C.

6. Samples has 1-4 g sodium chloride added, and were compared to sampleshaving 1-8 g potassium carbonate added. The salts were added to frozenblood, which was sealed in the serum vial and then allowed to thaw.

Shaking the samples had no effect on the quantity of acetaldehyde in thehead space. Heating samples to various temperatures, starting at 30° C.and increasing by 10° C. up to 100° C. produced approximately a ten-foldincrease in acetaldehyde quantity. Temperature effects on quantity ofblood head-space compounds are shown in FIG. 4a. Equilibrating samplesin the water bath produced only about a two-fold increase inacetaldehyde. Incubation time effects on blood head-space volatilecompounds are shown in FIG. 4b. Increasing the percentage of total SPFin the sealed vial between 33% and 90% caused a slight increase inacetaldehyde quantity. Adding or decreasing water to the blood had noeffect on acetaldehyde. Salting the blood with sodium chloride decreasedthe yield of acetaldehyde. Adding potassium carbonate produced markedincreases in acetaldehyde peaks. The effect of salting out withpotassium carbonate on blood head-space compounds is shown in FIG. 4c.Thus, heating the sample to higher temperatures for longer amounts oftime and the addition of potassium carbonate increased the quantity ofacetaldehyde present in the head space. The sample preparationprocedures differentially affected the yield of blood volatilecompounds. The analysis of humoral fluid volatile compounds in otherspecies will likely benefit from optimizing sample handling procedures.

To collect sufficient quantity of acetaldehyde for mass spectralidentification, potassium carbonate (K₂ CO₃) was added to the blood andthe blood was heated for 30 minutes at 90° C., as described above. Bloodhead-space gas was derivatized with 2,4-dinitrophenyl hydrazine (DNPH)according to the following procedures.

Frozen cow's blood (250 ml) was thawed in a water bath and 10 ml of theblood added to twelve 30 ml glass vials. The vials were capped andplaced in a -60° C. freezer for at least 1 h. The vial caps were removedand 8 g of K₂ CO₃ was added to each frozen sample. The vials wererecapped and refrozen.

Ten mg DNPH was placed in a 0.3 ml glass micro-reaction vial with arubber septum screw-top lid and dissolved in glacial acetic acid. Thevial was then placed in an ice-water slurry, and the slurry and vialplaced in a -60° C. freezer for at least 1 h. Three of the 30 ml vialscontaining bulk blood were removed from the freezer and allowed to thawat room temperature for 15 minutes, shaken by hand to mix the K² CO³ andblood, then placed in a 90° C. water bath for 30 minutes.

The micro-reaction vial was removed from the freezer and a 22 gaugeneedle pierced through the septum to allow a vent for excess pressure. Ablood sample was removed from the water bath and 5 ml of head spacesampled from it with a Hamilton Gas-Tight syringe, equipped with theThermo-Syringe (Reno, Nev.) set to 50° C. and a 22 gauge, 1 inch needle.The syringe's needle was then pierced through the septum on themicro-reaction vial and down far enough into the reaction mixture thatthe end of the needle entered into the excess sediment of DNPH on thebottom of the vial.

The syringe was then put into a Sage Instruments (Cambridge, Mass.)syringe pump. The pump was operated at 2.3 ml min⁻¹, and all thehead-space sample bubbled through the reaction mixture. Head space wassampled and injected into the DNPH three times from each of the threevials, and then the micro-reaction vial was put back into the freezer,and three more 30 ml vials of blood removed from the freezer forthawing. After the final three 30 ml vials had been sampled, themicro-reaction vial was put back in the freezer for 30 minutes. It wasthen removed and allow to thaw enough so that it could be removed fromthe ice around it, and then was allowed to warm to room temperature.

The reaction mixture was pulled into a 5 ml disposable syringe andpushed back through a 0.2 μm disposable syringe filter, with the liquidpart of the reaction mixture being collected. Two to three drops ofeither acetone or hexanal were added to the liquid, and the mixturesonicated for 5 minutes to react with whatever unreacted DNPH may havebeen present. Hexanal was used because it permitted a more rapidreaction with DNPH. The derivatives with DNPH, however, took too long toelute from the HPLC column, which was used to separate the derivatives.Acetone was therefore used in HPLC runs, and the derivatized peakscollected as separate fractions. An acetaldehyde-free control sample wasprocessed with the acetone procedure, which verified the absence ofmeasurable acetaldehyde contamination of the acetone.

Gas chromatography/mass spectrometry (GC/MS) analysis was performed on aHewlett-Packard Model 5970 GC/quadruple mass spectrometer coupled to anHP model 5890 GC fitted with an HP Ultra-1 cross-linked methyl siliconemicrobore capillary column (12.5 m, 0.30 mm O.D., 0.20 mm I.D.). Massspectral detector (MSD) ionization was by electron impact at 70 eV, ionsource (chamber) temperature was 220-250° C. The MSD was tuned (m/e 69,219 and 502) with perfluorotri-n-butylamine (PFTBA), normalized todecafluorotriphenylphosphine (DFTPP). The GC transfer line was held at280° C. The GC was equipped with an on-column injection system.

On-column injection was performed with a 10 μl Hamilton syringe fittedwith a 10 cm fused silica needle (O.D. 0.17 mm). The system wascontrolled by a HP Model 59970A work station. Conditions for on-columninjection and GC analysis were as follows: helium (head) pressure 5.5psi; detector 280° C.; initial column temperature held at 40° C. for 1minute, followed by ramping at 25° C. min⁻¹ to a final temperature of270° C. These conditions allowed clear separation of DNP derivatives ofacetone and acetaldehyde. To verify the effectiveness of derivatization,we used HPLC to show the presence of reacted products. HPLC conditionsincluded use of a C 18 reversed-phase column with methanol-water as theeluting solvent.

In some cows, there was also an early-eluting fifth peak. Mass spectraof blood head-space that was derivatized with DNPH sample and acomparably prepared acetaldehyde standard is shown in FIG. 5a. The 224molecular ion is the sum of the mass of DNP (198) and acetaldehyde (44)minus the mass of water, which is eliminated in the reaction. Thecomplete characteristic chromatographic profile appearing 1-3 days priorto estrus is shown in FIG. 5b.

Acetaldehyde peaks occurred in larger than normal amounts 1-3 days priorto "standing heat" or day of estrus. Since mating behavior occurs a fewhours before ovulation, the estrous specific compounds predict bothestrus and ovulation. The methods used with regard to bovines apply toany use in any mammalian species wherein one or more humoral volatilesare used to predict and detect estrus in animals or ovulation in humans.In humans, for example, the methods could be used for developing a"rhythm method" birth control diagnostic. The assay could also be usedto improve success rates with artificial insemination and embryotransfer, and with oocyte maturation procedures.

EXAMPLE II

Head-space gas chromatography analysis was performed on 10 ml milksamples following the analytical procedures described in Example I. Milksamples were collected in the morning from four cows beginning 6 to 8days prior to estrus. The results of head-space gas chromatographyanalysis to determine the quantity of acetaldehyde in the milk samplesare shown in FIGS. 6a-6d. The data is expressed as absolute area underthe curve.

There was a distinct rise and then fall in acetaldehyde quantities,compared to the baseline quantities, just prior to the day of observedbehavioral signs of estrus (Day 0). FIG. 6d shows a succession ofprogressively increasing acetaldehyde peaks prior to estrus. Thispattern was unusual, and was observed in only one animal during oneestrous cycle. The greatest increase, then decrease in acetaldehyde inthat animal, however, occurred just prior to estrus. Thus, monitoringmilk acetaldehyde levels would enable predicting the occurrence ofestrus and ovulation.

EXAMPLE III

Known chemical reagent tests may also be used to determine the amount ofacetaldehyde or other low molecular weight volatile compounds subject tovariation during the reproductive cycle present in breath, body cavityair, humoral fluids, or the air above humoral fluids, from humans andanimals. In another embodiment of the invention, air adsorbent tubes areused to trap the low molecular weight volatile compound found in bodycavity air from the vaginal (reproductive) cavity of cows. The airadsorption tubes (Supelco ORBO™-25) contained an adsorbent and a packingmesh (Supelpak 20N). The adsorbent, 2-(hydroxymethyl)piperidine, reactswith acetaldehyde to trap the acetaldehyde in the mesh for subsequentultraviolet spectroscopy analysis. Acetaldehyde reacted with2-(hydroxymethyl)piperidine is stable and non-volatile, and will absorbultraviolet light at different wavelengths than when unreacted; thus thequantity of acetaldehyde can be measured. Other commercially availableair adsorption tubes which may be used to collect the samples includeNAPH 226119 (SKC) and NAPH 22627 (SKC). Acetaldehyde adsorption tubescontaining DNPH or 1,3-cyclohexanedione are also available forcollecting samples containing acetaldehyde.

Vaginal air samples were collected from cows, beginning at approximatelyday 15 or 16 of the 21 d estrous cycle. In the preferred embodiment,samples were taken at 12 h intervals. In an alternate method, samplesmay be taken at 24 h intervals. Samples may be taken more frequently, ifdesired, to monitor the variations in the quantity of the low molecularwe

Vagimpound present in the sample.

Vaginal air samples were collected by removing the glass seals from anair adsorbent tube and fitting both ends of the tube with a flexiblehose. The free end of one of the flexible hoses was then connected to aportable pump having a flow meter calibrated to draw in 500 cc/min ofair, for a total collection of a 5 l sample. A 5 l air sample was foundto provide sufficient quantities of low molecular weight volatilecompound for analysis.

The free end of the second flexible hose was positioned near the vulvabut outside of the vaginal cavity of the animal. Because of the volatilenature of acetaldehyde, air collected from outside of the animal'svaginal cavity contained sufficient quantities of acetaldehyde formeasuring. The low molecular weight compound collected will be from thereproductive tract air and the vulval gland secretions. Secretions fromall skin glands would contain some quantity of acetaldehyde, however,since all body fluid compartments are in equilibrium.

Once the air sample was drawn through the air adsorption tube, the tubewas sealed at each end by removing the flexible hoses and capping eachend of the tube. The tubes containing the collected sample are frozen invapor nitrogen or according to recommended procedures for the handlingof the air adsorbent tube. The quantity of low molecular weight compoundwas analyzed using standard ultraviolet spectroscopy techniques.

FIG. 7 graphs the quantity of acetaldehyde in vaginal air samplesobtained from three cows: Cow #1, Cow #2 and Cow #3, respectively.Sampling began during the proestrus phase of each animal's estrouscycle. Samples were taken every 24 h from cow #1, at 12 h intervals fromcow #2, and at alternating 1 time/d and twice/d from cow #3 during thecollection period to monitor the quantity of acetaldehyde present in thevaginal air. The sample size was five l of vaginal air, collected at 500cc/min. Each sample was collected from outside the body of the animal atthe vulvar area, as described herein.

Vaginal air acetaldehyde peaks are shown to vary greatly with day ofsampling. However, the peak acetaldehyde quantity followed by a rapidand sharp decline in acetaldehyde in vaginal air consistently occurredat or near estrus. Estrus in all three cows was confirmed by visualobservations of standing mating behavior and metestrous bleeding 3-4days after the drop in acetaldehyde.

The significant quantities of acetaldehyde in vaginal air is correlatedwith the stages of the estrous cycle. Observations of acetaldehydespikes in vaginal air will allow the prediction of estrus and/orovulation in animals and humans.

Since there are extremely dramatic variations in the amount ofindividual volatile compounds present in body constituents, anytechnique for measuring the quantity of the compound present, used atspaced apart intervals throughout the cycle will produce an indicationof the quantity of the compound present at the time of sampling.

In the alternative, body cavity air samples may be obtained from withinthe body cavity. For example, the free end of the a tube connected tothe air adsorption tube described above may be inserted into the vaginaor cervix of a cow and an air sample drawn through the tube. Althoughthis method may be used to obtain samples for monitoring the variationsin the quantity of low molecular weight compound in the body cavity air,it is not as desirable from the standpoint that it will require morehandling of the animal, resulting in greater stress on the animal. Thecollection of the sample will also have to be done more slowly so as notto risk collapsing the body cavity as the sample is drawn.

In yet another embodiment of the invention, body cavity air,particularly vaginal (reproductive) cavity air, may be collected usingthe kit shown in FIG. 8. A vulva of a cow is shown generally at 800. Anair adsorption tube 802 of the type described above in this example ispositioned in a cannula 804. The cannula 804 may be made from anysubstance, such as plastics, which will not irritate the animal. Thewall of the cannula may be of any thickness which will not irritate theanimal and must have an internal diameter suitable to accommodate theair adsorption tube 802. Preferred dimensions for the cannula are 20 cmin length and 2 cm outer diameter with a 2 mm thick wall. A first end806 of the cannula will have a plurality of holes 808. A second end 810of the cannula will be sealed at lid 812. A first tubing 814 will beconnected to air adsorption tube 802, and exit lid 812 through a firstorifice 816. Tubing 814 is connected to a metered hand held pump 818.Tubing 814 may be a tygon tubing or other suitable tubing for connectingthe air adsorption tube to the pump. A second tubing 822 is connected topump 818 and enters the cannula through second orifice 820 in lid 812.The cannula containing the air adsorption tube is placed approximately10 cm into the vagina of the animal. Activation of the pump 818 drawsbody cavity air from the body cavity through holes 808 into the cannulaand through air adsorption tube 802. The flow of body cavity air isshown by arrows a, b, c, and d. Once the air is drawn through the airadsorption tube, it passes through tubing 814, through pump 818 and thenforced through tubing 822 back into the cannula. The flow of air backinto the cannula will facilitate the movement of air into the cannula atholes 808. Body cavity air samples should be taken slowly so as not torapidly evacuate the air and collapse the cavity. Drawing 500 cc/min airfor a total sample of 5 l of cavity air is preferred. However, lesssample may be collected to monitor acetaldehyde quantities present sincebody cavity air samples taken from inside the animal will not beinfluenced by barn or environment air, as with samples drawn fromoutside the animal.

EXAMPLE IV

Humoral fluid samples may also be analyzed using batch mode analyses,wherein a reagent which reacts with the low molecular weight compoundsubject to variation during the reproductive cycle being measured isadded to a humoral fluid sample. The sample is then processed to purifyand quantify the reactant using standard spectroscopic or fluorometrictechniques.

Acetaldehyde quantities in milk samples collected during the estrouscycle of cows have been measured and monitored to determine the phase ofthe mammal's reproductive cycle and to predict ovulation and estrus.Quantities of acetaldehyde in the samples were measured using thefollowing described procedures.

A 25 ml milk sample is collected and 2 ml saturated sodium chloride(NaCl) and 20 ml hexane added. The milk, NaCl and hexane mixture isshaken and allowed to stand for 5 min. The hexane layer separates andwill contain milk lipids. The top layer of hexane and lipids is pipettedoff and discarded. Proteins are precipitated from the remaining sampleby adding 7 ml of 3M potassium carbonate (K₂ CO₃) and centrifuging 5 minat 2000 g. The supernatant is removed and 2.5 ml DNPH solution (1 mg/mlDNPH in 6M HCl) added. The sample is then heated on a shaker at 60° C.for 10 min for the reaction to occur. The sample is filtered usingstandard filter paper and 2 ml of filtrate slowly loaded into a C18solid-phase extraction cartridge. The C18 cartridge is rinsed 1 min withwater, followed by 1 min with dilute acid (1% HCl). TheDNPH-acetaldehyde reactant diluted with 2 ml acetonitrile. This steptakes approximately 3 min. A 20 μl sample of the DNPH-acetaldehydeeluate is injected into an HPLC and read at UV wavelength 360.

In alternate methods, 7 ml of 3M perchloric acid may be added instead of7 ml K₂ CO₃ to precipitate proteins. As another alternative,1,3-cyclohexanedione solution may be added instead of DNPH solution.Other batch mode analyses of samples for quantifying the quantity ofacetaldehyde in a humoral fluid sample will be known to those skilled inthe art.

Batch processing milk and other humoral fluid samples to monitorvariations in the quantity of acetaldehyde or other low molecular weightvolatile compound subject to variation during the reproductive cycle caneasily be used to monitor reproductive cycles in humans and animals.

EXAMPLE V

Air collected from above a humoral fluid may be analyzed to measure thequantity of a low molecular weight volatile compound subject tovariation during the reproductive cycle. A kit for sampling the airabove humoral fluids, such as milk and blood, is shown in FIG. 9. Ahumoral fluid sample 900 is placed in a container 902, which is thenplaced in a sonication bath 904. An air pump 906 is used to force airthrough tubing 908 and into the sample 900, thereby increasing theamount of low molecular weight volatile compound moving out of thehumoral fluid. The sonication bath further facilitates flushing the lowmolecular weight volatile compound out of the humoral fluid and into theair above the fluid. It is preferred that the container be sealed with alid 916, with tubing 908 and an entry tube 910 passing through lid 916at a first orifice 918 and a second orifice 920, respectively. Sealingthe container will reduce the escape of the low molecular weightvolatile compound.

The air above the humoral fluid is drawn into entry tube 910 and throughan air adsorption tube 912 by pump 906. The low molecular weightvolatile compound present in the air above the humoral fluid will betrapped in the air adsorption tube 912. The air adsorption tube 912 maybe any of the types described in Example III herein. Exit tube 914connects the air adsorption tube to the pump. The flow of air from thepump into the humoral fluid and back into the pump is shown by arrows a,b, c, and d.

It is preferred that a 5 l sample of air above the humoral fluid betaken. The low molecular weight volatile compound trapped in the airadsorption tube may be analyzed using the procedures described inExample III herein. A larger or smaller volume may be taken, however, ifadequate for quantifying the low molecular weight volatile compoundusing analytical techniques known to those in the art.

EXAMPLE VI

Low molecular weight volatile compounds subject to variation during thereproductive cycle may be measured by commercially availableelectrochemical detectors. An electrochemical detector capable ofdetecting and measuring a selected low molecular weight volatilecompound, such as acetaldehyde, can be attached to the tail area of acow. The electrochemical detector will measure the quantity of the lowmolecular weight volatile compound in the air above skin glandsecretions in the area of the vulva as well as air emitting form thereproductive cavity or vagina. The detector may be designed to have anaudio or visual means for signalling a change, or alerting a herdsmanthat the quantities of acetaldehyde, or other low molecular weightvolatile compound present in body cavity air and/or in air above skingland secretions has reached a particular level indicative of the peakobserved near the time of estrus and/or ovulation.

EXAMPLE VII

Low molecular weight volatile compounds subject to variation during thereproductive cycle may also be monitored by measuring the quantity ofthe compound in a sample obtained from the mouth of a mammal. Thismethod would be preferred for use in predicting ovulation in humans. Anoral collection pad may be used to collect a sample of oral fluid withincreased concentrations of serum analytes. The swab would then beanalyzed to determine the quantity of low molecular weight volatilecompound, such as acetaldehyde, in the sample. Samples would be taken amultiple number of times, beginning prior to ovulation, in order tomonitor the variations in the quantity of compound present in eachsample. A sharp increase in acetaldehyde, or other compound beingmonitored, followed by a sharp decline in the amount of the compound,will enable predicting the occurrence of ovulation.

The examples included are not intended to limit the scope of the presentinvention. Other substitutions, modifications and variations areapparent to those skilled in the art without departing from thedisclosure and scope of the invention.

What is claimed is:
 1. A kit for predicting the occurrence of ovulationin a mammal comprising:sample collection means for non-invasivelycollecting a body constituent sample containing acetaldehyde; a detectorfor measuring the quantity of the acetaldehyde in the sample; and meansfor monitoring variations in the amount of the acetaldehyde in thesample over said mammal's reproductive cycle and comparing thosevariations with variations that are known to occur over that cycle. 2.The kit of claim 1, wherein the detector is selected from the groupconsisting of gas chromatographs, ultraviolet-visible spectrometers, andelectro-chemical detectors.
 3. The kit of claim 1 wherein the samplecollection means is an adsorption vessel containing an adsorbent withwhich acetaldehyde will react.
 4. The kit of claim 3, wherein theadsorbent is selected from the group consisting of1-(hydroxymethyl)piperidine, dinitrophenylhydrazine and1,3-cyclohexanedione.
 5. The kit of claim 1, wherein the samplecollection means is a device for collecting a gas sample.
 6. The kit ofclaim 1 further comprising a pump connected to the sample collectionmeans for drawing a sample into the sample collection means.
 7. The kitof claim 1, wherein the monitoring means generates a signal when thevariation in the amount of acetaldehyde in the sample is a variationthat is indicative of the onset of ovulation.
 8. A kit for predictingthe occurrence of ovulation in a mammal comprising:sample collectionmeans for non-invasively collecting a body constituent sample containingacetaldehyde; a detector for measuring the amount of the acetaldehyde inthe sample; and means for comparing the amount of acetaldehyde detectedin the sample with amounts that are known to occur over that cycle andgenerating a signal when the detected amount of acetaldehyde isindicative of the onset of ovulation.